棉铃虫(Helicovera armigera.Hübner)是一种世界性农业害虫，其抗药性机制一直是该领域研究的前沿和热点问题。羧酸酯酶是其代谢抗性相关的主要解毒酶系之一。课题组前期对棉铃虫系列羧酸酯酶研究发现，其对有机磷杀虫剂有解毒活性；当CarE001C的127位由丙氨酸（Ala）突变为天冬氨酸(Asp）后，活性提高7倍。在此基础上，本项目拟采用饱和突变技术，构建CarE001C氧阴离子洞126位和127位的突变体库，通过分子定向进化和酶学分析方法研究突变对酶促动力学特性的影响机理。本研究具有以下目的和意义：1）解析棉铃虫羧酸酯酶氧阴离子洞126位和127位氨基酸组成（结构）与酯酶催化活性的关系，为新型农药设计提供理论指导；2）分析和预测棉铃虫羧酸酯酶在抗性发展中可能出现的分子机制，为棉铃虫综合防治提供实践指导；3）为开发昆虫解毒酶羧酸酯酶应用于环境修复奠定前期基础。

The cotton bollworm, Helicoverpa armigera (Hübner), is a major pest of many agricultural crops around the world, the mechanism of insecticide resistance is always the leading research and hot issues in recent years. Carboxylesterases(CarEs) is the major class of detoxification enzymes involved in the development of insecticide resisitance in insects. Our previous study on CarEs from Helicoverpa armigera showed that esterases had some low activities toward organophosphates (OPs). A mutation 001CA127D has shown increasing 7-folds of the hydrolysis activity of diethyl-phosphates than that of the wild type enzyme. In this research proposal, we are investigating the mutations at the oxyanion hole of CarE001C and enhanced OP hydrolase activity. Mutant libraries of both G126 and A127 positions will be produced by saturation mutagenesis technology. The hydrolysis activities of all mutations will be screened and the kinetic properties of the mutants will be analyzed. The results will provide information for understanding the OP resistance mechanism at the enzymatic level. The aims and significance of this study are: 1) to elucidate the relationship between the amino acids of oxyanion hole of CarE001C and its catalytic function and will be used for rational designing and developing new kind of insecticides; 2) to provide useful data for understanding the molecular mechanism of the development of metabolic resistance to insecticides and benefit to the Integrated Pest Management (IPM) in agriculture; 3) to develop OP degrading enzymes for bioremediation.